[Advances in Marine Biology] Aquatic Geomicrobiology Volume 48 || The Nitrogen Cycle

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  • 8.2. Nitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

    8.3. Nitrogen fixation and assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2661. INTRODUCTIONThe Nitrogen Cycle

    1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

    2. The Global Nitrogen Cycle and Human Perturbations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

    3. Biological Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

    3.1. The nitrogenase enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

    3.2. Ammonium assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

    3.3. The oxygen problem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

    3.4. Phylogeny of nitrogen-fixing organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

    3.5. Nitrogen fixation in aquatic environments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

    4. Microbial Ammonification and Nitrogen Assimilation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

    4.1. Ammonification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

    4.2. Deamination and ammonium incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

    4.3. Nitrogen mobilization and immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

    4.4. Anaerobic nitrogen mineralization and ammonium behavior in sediments . . . . . . 225

    4.5. New versus regenerated nitrogen in pelagic ecosystems. . . . . . . . . . . . . . . . . 230

    5. Nitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

    5.1. Biochemistry and thermodynamics of nitrification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

    5.2. Phylogeny of chemolithoautotrophic nitrifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

    5.3. Environmental factors aVecting nitrification rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

    6. Dissimilatory Nitrate Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

    6.1. Biochemistry of denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

    6.2. Biochemistry of NO3 ammonification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2516.3. Phylogeny and detection of denitrifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

    6.4. Environmental factors aVecting denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

    7. Anammox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

    8. Isotope Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

    8.1. Denitrification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265Chapter 7Nitrogen is a key constituent of many important biomolecules, such as

    amino acids, nucleic acids, chlorophylls, amino sugars and their polymers,

    and it is essential to all living organisms. Nitrogen has the property of an

    ADVANCES IN MARINE BIOLOGY VOL 48 2005 Elsevier Inc.0-12-026147-2 All rights reserved

  • 206 CANFIELD ET AL.Figure 7.1 The microbial nitrogen cycle with an indication of the valence state ofthe various nitrogen-containing compounds involved.eight-electron diVerence between its most oxidized and reduced compounds(Figure 7.1). Thus, the redox cycling between nitrogen compounds forms

    the basis for numerous microbial metabolisms. Many of these microbial

    processes, in turn, control the availability of nitrogen in the environment

    and hence are significant in regulating the activities of primary producing

    eukaryotes, which require a ready source of nitrogen for growth.

    In a quick survey of the various microbial nitrogen-transforming process-

    es, we start with N fixation. In this process, prokaryotes transform atmo-

    spheric dinitrogen (N2) into ammonia (NH3), a biologically available form

    that can be incorporated into biomolecules (Sprent and Sprent, 1990). The

    diYculty in N fixation is breaking the strong triple bond holding the twoN atoms in N2 together. Most organic nitrogen is recycled into inorganic

    form by a process known as ammonification in which nitrogen-containing

    biomolecules are degraded by microorganisms or digested by animals. The

    released ammonium (NH4 ) can either be re-assimilated by microbes orplants and transformed into new biomolecules, or it can be oxidized by an

    assemblage of largely chemolithoautotrophic prokaryotes. The resulting

    oxidized nitrogen forms, nitrite (NO2 ) and nitrate (NO3 ), can either be

    assimilated by microorganisms and plants or be denitrified to N2 by a

    number of heterotrophic prokaryotes using oxidized nitrogen as electron

    acceptor and organic carbon, for example, as electron donor. The resulting

  • N2 is returned to the large atmospheric pool and thus, on short term, is lost

    for further biological transformations. Most bioavailable nitrogen is recycled

    several times between autotrophic and heterotrophic organisms, because the

    rates of nitrogen input to the biosphere by N fixation, and output by denitri-

    fication, are at least an order of magnitude slower than the internal cycling

    rates. This conventional view of the nitrogen cycle has recently been amended

    with the anammox process, in which ammonium oxidation is coupled to

    nitrite reduction, leading to the production of N2. This process may be of

    significance in the nitrogen cycle of aquatic enviroments.

    In this Chapter we explore how the interplay between microbial processes

    and the geochemical environment controls the cycling of nitrogen in aquatic

    ammonium substituted within potassium-rich minerals (Table 7.1) (Krohn

    THE NITROGEN CYCLE 207et al., 1988). Rock weathering liberates this nitrogen, which then becomes

    available to living organisms. Nitrogen in sediments and sedimentary rocks

    is the next largest pool. Here, too, nitrogen is mostly fixed as ammonium in

    secondary silicate minerals (Blackburn, 1983), and this nitrogen source also

    becomes available during weathering. Of comparable size is the reservoir of

    atmospheric N2, which accounts for 78% of the gas in the atmosphere. The

    Table 7.1 Major nitrogen reservoirs on earth

    Type of pool Location Pool size (g N)

    N2 gas Atmosphere 3.8 1021Living biomass (LPON) Aquatic and terrestrial 1.3 1016Dead organics (DPON + DON) Aquatic and terrestrial 9.0 1017Inorganic (NH4 , NO

    2 , NO

    3 ) Aquatic and terrestrial 2.4 1017

    Inorganic (fixed NH4 ) Sediments and sedimentary rock 4.0 1021Inorganic (NH4 within minerals) Igneous rock 1.4 1022

    From Delwiche, 1970; Blackburn, 1983; Madigan et al., 2002.The largest reservoir of nitrogen at the Earths surface, including the crust

    and the atmosphere, is in igneous rocks, where nitrogen is primarily found asenvironments. Thus, we look at how various microbial pathways promote

    the transformation of nitrogen compounds and how environmental factors

    regulate the operation and intensity of these pathways. In addition, we also

    explore how nitrogen isotopes are fractionated during microbial transfor-

    mation. First, however, we consider aspects of the global nitrogen cycle and

    the influence of human activities.

    2. THE GLOBAL NITROGEN CYCLE AND HUMANPERTURBATIONS

  • biologically available forms of fixed nitrogen mainly consist of dissolved

    NH4 , NO2 , and NO

    3 in aquatic and terrestrial environments, but this pool

    is small compared with the atmospheric reservoir (0.006%) and the reservoir

    comprising dead organic detritus (25%) (Vitousek et al., 1997). Livingbiomass is the smallest nitrogen reservoir, only about 1% of the size of the

    dead organic detrital pool.

    Pool sizes tend to be inversely related to their biological importance. The

    large igneous and sedimentary pools, for example, are not actively cycled by

    organisms, although rock weathering can contribute a locally significant

    source of nitrate to surface and ground waters (Holloway et al., 2001). The

    biological processes of N fixation and denitrification interact with

    the large atmospheric N2 pool (Figure 7.2), but even here, the turnover

    time of the atmospheric N2 pool is slow. The inorganic ions, NH4 , NO

    2 ,

    and NO3 , are distributed in aqueous solution throughout the ecosphere, andthey form small actively cycled reservoirs. Assimilation, mineralization,

    208 CANFIELD ET AL.Figure 7.2 The global nitrogen cycle with an indication of the most importanttransfer processes. Rates are given as 1012 g N yr1. NX indicates inorganic com-bined nitrogen. Based on data from Roswall (1983); Codispoti and Christensen(1985); Seitzinger and Giblin (1996); Vitousek et al. (1997); Seitzinger and Kroeze(1998). Other recent estimates of marine dentrification are as high as 450 Tg Ny1

    indicating a large possible imbalance of the marine N budget (Codispoti et al., 2001).

  • (Gruber and Sarmiento, 1997). Industrial nitrogen fixation into synthetic

    obvious that humans have heavily impacted the global nitrogen cycle. An-

    THE NITROGEN CYCLE 209other example of this impact comes from the cycle of nitric oxide (NO),

    which can be transformed in the atmosphere into nitric acid, a major

    component of acid rain (Vitousek et al., 1997). Fossil fuel burning emits

    more than 20 1012 g N yr1 as NO, while deforestation through burningcontributes another 10 1012 g N yr1 as NO. Furthermore, a substantialfraction of the total of 5 to 20 1012 g yr1 of NO nitrogen emitted fromsoils is human related. Overall, 80% or more of NO emissions worldwide are

    generated by human activities.

    To consider the ammonia cycle, nearly 70% of the global emissions to

    the atmosphere are human related. Ammonia volatilization from fertilized

    fields contributes an estimated 10 1012 g N yr1, release from domesticanimal wastes liberates about 32 1012 g N yr1, and forest burning con-tributes a further 5 1012 g N yr1. Synthetic nitrogen fertilizer input hasincreased fourfold over the last four decades (about 20 1012 g N yr1 in1960) and is expected to rise from a current level of 80 1012 g N yr1 to 1341012 g N yr1 in 2020 (Vitousek et al., 1997). Additional non-biologicalsources of fixed nitrogen to the Earths surface include volcanic outgasing

    and atmospheric fixation through ionizing radiation and electrical discharge.

    3. BIOLOGICAL NITROGEN FIXATION

    Only specialized prokaryotes contain the enzyme nitrogenase and the ability

    to fix N2 into a biologically useful combined form (Sprent and Sprent, 1990).

    These organisms, known as diazotrophs, are found in both prokaryote

    domains. They may be found both free-living and in symbiotic association

    with plants. Nitrogen-fixing prokaryotes utilize a wide variety of diVerentenergy metabolisms, including oxygenic photosynthesis, sulfate reduction,

    methanogenesis, anoxygenic photosynthesis, and chemolithoautotrophy

    (Table 7.2).fertilizer from the Haber-Bosch process produces roughly 80 1012 g yr1 offixed nitrogen, similar in magnitude to marine biological N fixation. It isand nitrification (internal cycles on land and in the ocean as shown in Figure

    7.2) are quantitatively the most important processes linking the inorganic

    reservoir with the likewise small and actively cycled reservoirs of living and

    dead organic nitrogen (JaVe, 2000).Biological nitrogen fixation on land amounts to about 180 1012 g yr1,

    which includes about 40 1012 g yr1 from agriculturally managed legumecrops (Figure 7.2). This rate exceeds rates of biological nitrogen fixation in

    the marine environment, with an estimate of about 100 1012 g yr1

  • Table 7.2 Selected list of free-living microbial genera, which contain N-fixingspecies or strains. The list is separated into diVerent metabolic types of microorgan-

    210 CANFIELD ET AL.isms with indication of their preferred habitat

    Metabolism Genus or type Environment

    Aerobes Azotobacter Sediment/waterFacultative anaerobes

    (not fixing when aerobic)Klebsiella Sediment/waterPaenibacillus Microbial mat/rhizosphereEnterobacter Sediment/animal gutEscherichia Animal gut

    Microaerophiles(when fixing N2)

    Xanthobacter Microbial mat/sedimentThiobacillus Microbial mat/sedimentAzospirillum RhizosphereAquaspirillum Water

    Anaerobes Clostridium SedimentDesulfovibrio SedimentMethanosarcina SedimentCombined, a major nutrient for plant primary production, is generally

    found in short supply in marine areas, where significant loss of occurs

    through denitrification (see Section 6). Nitrogen is also lost through burial

    as organic N and as adsorbed and mineral-bound ammonium in sediments

    (Figure 7.3). Nitrogen fixation is therefore needed to replace lost nitrogen.

    The overall process of N fixation is exergonic at standard conditions (Equa-

    tion 7.1), but the great stability of the NN bond in N2 makes it extremelyunreactive at room temperature.

    3H2 N2 ! 2NH3; DG 0 33:4 kJ mol1 7:1Indeed, in the industrial Haber-Bosch process this reaction will only occur

    when operated at temperatures between 300 and 4008C and at pressures

    Methanococcus SedimentPhototrophs Chromatium Microbial mat/sediment

    Chlorobium Microbial mat/sedimentThiopedia Microbial mat/sedimentRhodospirillum Microbial mat/sedimentRhodopseudomonas SedimentOscillatoria Microbial mat/waterNodularia WaterAnabaena WaterNostoc Microbial matCalothrix Microbial matGloeotheca Microbial mat

    Modified from Capone (1988) and Postgate (1998).

  • THE NITROGEN CYCLE 211between 35 and 100 MPa. Prokaryotes outperform the industrial pro-

    cess with the nitrogenase enzyme complex. Thus, nitrogenase reduces the

    triple-bonded NN molecule to NH4 at normal environmental tempera-tures and pressures, but it is not completely N2 specific, as other triple-

    bonded molecules such as acetylene (HCCH) and hydrogen cyanide(HCN) are also reduced.

    Figure 7.3 Microbial nitrogen cycling in aquatic environments showing themajor transformations within (uppercase letters) and between (lowercase letters)anoxic sediment, oxic sediment, the water column, and the atmosphere. A, nitrogenfixation; B, NOx assimilation; C, ammonification; D, NH

    4 assimilation; E, NH

    4

    oxid...

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